Isolation and characterization of a fiber-specific β-tubulin promoter from cotton

ABSTRACT

The present invention relates to a cotton β-tubulin gene CFTUB2, and active fragments thereof. These promoters show strong fiber-specific activity.

TECHNICAL FIELD

The present invention relates to the field of plant molecular biology,in particular to transgenic plants and promoters useful in creatingtransgenic plants, and more particularly to fiber-specific promoters.

BACKGROUND OF THE INVENTION

Cotton is the most extensively used natural fiber in the textileindustry. Annual production of cotton worldwide is over 100 millionbales valued at 45 billion U.S. dollars. Although significantimprovements have been made in quality and yield of the fibers by meansof classical breeding in the past decades, the potential for furtherimproving fiber properties through classical breeding is limited due torequirements for species compatibility and available traits. Geneticengineering provides novel approaches for further improving cotton byintroducing genes to create new germplasms with highly desirablecharacteristics.

Cotton fibers (seed hairs) are single-cell trichomes that undergo rapidand synchronous elongation. Cortical microtubules provide spatialinformation necessary for the alignment of cellulose microfibrils thatconfine and regulate cell elongation [Giddings and Staehelin, 1991; Cyrand Palevitz, 1995; Fisher and Cyr, 1995]. Fiber development consists offour overlapping stages (i.e. initiation, primary cell wall formation,secondary cell wall formation and maturation) [Basra and Malik, 1984].Tubulins and actins may play functionally important roles in developingfiber cells. Mature fiber is a biological composite of cellulose, water,small quantities of proteins, pectins, hemicellulose, mineralsubstances, wax, small amounts of organic acids, sugars, and pigmentsthat provides excellent wearability and aesthetics [Arthur, 1990; Basraand Malik, 1984; Ryser, 1985]. Many genes are required for the fiberdifferentiation and development. These genes are differentiallyexpressed during different stages of the fiber development, and so faronly a few of the genes involved in the biosynthesis of the largenumbers of fiber-specific structural proteins, enzymes, polysaccharides,waxes or lignins have been identified [John and Crow, 1992; John, 1996a;Song and Allen, 1997; Ma et al., 1997; Kawai et al., 1998; Whittaker andTriplett, 1999]. These isolated genes may be considered as havingpotential application in cotton fiber improvement due to the characterof their fiber-specific expression. For example, John has been usingfiber-specific gene promoters to produce genetically engineering cottonfor altered fibers [John, 1996b, 1997a, 1997b].

A promoter is a DNA fragment that determines temporal and spatialspecificity of gene expression during plant and animal development. Manytissue-specific genes and their promoters were identified and isolatedfrom a wide variety of plants and animals over the past decade,including some cotton tissue-specific genes and promoters (Loguerico etal., 1999; Kawai et al., 1998; Song and Allen, 1997; Ma et al, 1997;John, 1996a; Rinehart et al., 1996; Hasenfratz et al, 1995; John andPeterson, 1994; John and Crow, 1992). A few promoters have been shown tocontrol gene expression in a fiber-specific manner in cotton (Rinehartet al., 1996; John, 1996a; John and Crow, 1992). Some planttissue-specific promoters can be utilized to express foreign proteins inspecific tissues in a developmentally regulated pattern [John, 1996b,1997a, 1997b].

SUMMARY OF THE INVENTION

A fiber-specific gene (named CFTUB2), encoding β-tubulin, was isolatedfrom cotton. The isolated complete CFTUB2 cDNA is 1.623 kb in lengthincluding 1.338 kb of open reading frame. Based on the CFTUB2 cDNAsequence, two CFTUB2 promoter fragments (1.433 kb and 0.984 kb) wereisolated from cotton. The two CFTUB2 promoter fragments (1.3 and 0.9 kb)were fused with the GUS gene to construct gene expression vectors foranalyzing the function of the promoter. Transgenic cotton and tobaccoplants with the CFTUB2 promoter/GUS fusion genes were identified bySouthern blot hybridization. In all the transgenic cotton plantsstudied, GUS activity was detected only in young fibers, but not in theflower organs such as anthers, petals and sepals, or in leaves androots. This result, together with Northern blot analysis, indicates thatthe CFTUB2 promoter is fiber-specific in cotton. The promoter controlsspecific gene expression at the transcriptional level in cotton fibers.The isolated promoter may be used in improving cotton fibers to createnew cotton varieties with high fiber quality and yield by genemanipulation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide sequence of the cotton CFTUB2 gene cDNA(1623 bp; SEQ ID NO: 1).

FIG. 2 shows the nucleotide sequence of the isolated 1433 bp CFTUB2promoter fragment (SEQ ID NO: 2. The 984 bp fragment corresponds tonucleotides 449–1433 of this sequence.

FIG. 3 shows constructs of the CFTUB2 promoter fused with the gus genein expression vectors.

DETAILED DESCRIPTION

The CFTUB2 promoter is an active fiber-specific promoter in cotton.Results of a Northern blot analysis of cDNAs from a variety of cottontissues showed that a cDNA clone comprising the CFTUB2 gene was stronglyexpressed in young fibers of 8 and 14 days postanthesis (DPA), and alsoexpressed in young ovules of 4, 8 and 14 DPA, but less or not at all inother tissues. Sequencing of the cDNA clone revealed that it was 1623 bpin length containing a open reading frame of 1338 bp (FIG. 1). Comparingthe nucleotide and predicted polypeptide sequences of the cotton CFTUB2with the data banks, it was found that the CFTUB2 cDNA shared 96%–98%homology at the amino acid level and over 78% homology at the nucleotidelevel with the known β-tubulin cDNAs and genes from other plants (suchas Arabidopsis, tobacco, rice, soybean, maize, potato, carrot, etc.)[Liaud et al., 1992; Snustad et al., 1992; Villemur et al, 1994;Tonoike, et al., 1994; Taylor et al., 1994; Kang et al., 1994; Okamuraet al., 1997; Chu et al., 1998; Okamura et al., 1999].

The transcripts of the CFTUB2 gene exhibited the highest accumulation incotton young fibers of 8 DPA, and then there was a visible decrease inthe accumulation of the gene products (mRNA) with further development ofthe fibers. Comparison of gene expression in different developmentalstages of cotton ovules also showed that the gene transcriptsaccumulated more in 8 DPA ovules than in 4 and 14 DPA, and there was agradual and visible decrease to an undetectable level in theaccumulation of gene products with fiber development from 8 DPA to 28DPA. This suggests that the gene is specifically expressed with a strictregulation at the transcriptional level during cotton fiber and ovuledevelopment, as with other cotton fiber-specific genes [Whittaker andTriplett, 1999; Shin and Brown, 1999; Kawai et al., 1998; John, 1996a;Song and Allen, 1997; Ma et al, 1997; Rinehart et al., 1996; John andCrow, 1992].

Two fragments in the promoter region were isolated and cloned intopGEM-T vector, respectively. One fragment of the CFTUB2 promoter was1433 bp in length (FIG. 2), and another was 984 bp long. Both fragmentsfunctioned as active, fiber-specific promoters. The constructs of CFTUB2promoter/GUS fusion gene were used to transform tobacco and cotton byAgrobacterium-mediated gene transfer, using the pBI121 vector containingCaMV35S promoter/GUS fusion as a positive control. Consistent with theresults from Northern blot analysis, the GUS gene driven by CFTUB2promoter specifically expressed in the young fibers, but not in othertissues, in all the 31 transgenic cotton plants studied, while the GUSactivity was detected in all the tissues of positive control cottonplants (35S:GUS). A total of 36 transformed cotton plants were obtainedand transplanted in soil to grow to maturation. Similarly, it was foundthat under the CFTUB2 promoter, GUS gene activity was only detected inthe seeds in all of the 15 transgenic tobacco plants studied, suggestingthe CFTUB2 promoter activity was also tissue-specific in tobacco (thecotton fiber, being an elongated hair of the seed coat, findshistological correspondence in the tobacco seed coat). This result,together with the above Northern blot analysis, indicates that theCFTUB2 promoter controls gene specific expression at the transcriptionallevel in cotton fibers.

Accordingly, one embodiment of the present invention is a fiber-specificpromoter obtained from the cotton fiber β-tubulin gene CFTUB2.

Another embodiment of the present invention is a fiber-specific promotercomprising a 1433 kb active fragment of the cotton fiber CFTUB2 genepromoter.

Another embodiment of the present invention is a fiber-specific promotercomprising a 984 kb active fragment of the cotton fiber CFTUB2 genepromoter.

Stll another embodiment of the present invention is a promoter that iscotton fiber-specific comprising an active fragment of the CFTUB2promoter fragment of SEQ ID NO: 2. An active fragment is a sequence ofshorter length than SEQ ID NO: 2 which still retains activity as afiber-specific promoter in cotton. A fragment can comprise excisions,deletions, truncations or substitutions of the sequence of SEQ ID NO: 2,or a combination of these. A preferred active fragment is the fragmentconsisting of nucleotides 449–1433 of SEQ ID NO: 2.

The promoters of the present invention are useful in creating transgeniccotton having altered fiber characteristics. The use of thefiber-specific promoters of the present invention permits selectiveexpression of a transgene in the cotton fiber, permitting greaterlatitude in the types of transgenes employed. Selective expressionavoids problems such as the metabolic burden imposed on a transgenicplant by systemic expression of a transgene, or the adverse effects ofthe expression of a transgene in non-fiber tissues. Examples forexpressing desirable genes in cotton fiber, but not in other parts ofthe cotton plants include: (1) anthocynin genes for colored cotton, (2)silk protein genes from silk worm or spiders for increased strength ofcotton fiber, (3) and biosynthesis of polyhydroxybutrate in cotton fiberfor improved thermal properties and insulating characteristics [John, etal., 1996]. There are numerous examples in the art of fiber-enhancinggenes that could be advantageously linked to the promoters of thepresent invention, and used to transform cotton using well-knowntechniques (see, e.g., Umbeck, 1992), to achieve expression of thetransgene in transgenic cotton fibers. See e.g., John, 1996b, 1997a,1997b; John et al., 1996.

EXAMPLE 1 Isolation of Fiber-Specific cDNA Encoding CFTUB2 SequencesExpressed Early During Fiber Development of Cotton

Cotton seeds were surface-sterilized with 70% ethanol for 30–60 secondsand 10% H₂O₂ for 30–60 minutes, followed by washing with sterile water.The seeds germinated on ½ MS medium on light at 28° C. in a cultureroom, and cotyledons and hypocotyls cut from sterile seedlings were usedas transformation explant materials. Cotton plants were grown in potsfor DNA and RNA extraction.

Total RNA was extracted from young fibers, ovaries, anthers, petals,sepals, leaves and roots of cotton by using the guanidinium thiocyanatemethod or SV Total RNA Isolation System (Promega). Poly(A)+ RNA waspurified by using oligo(dT)-cellulose spin columns from an mRNApurification kit (Pharmacia Biotech). Cotton cDNA was synthesized byusing a cDNA synthesis kit (Pharmacia Biotech). Cotton cDNA librarieswere constructed by inserting the cDNA fragments into the ZAP expressvector (Stratagene).

Poly(A)+ RNAs from cotton young fibers of about 8 and 14 dayspostanthesis (DPA), respectively, were converted to cDNAs which wereused to construct cotton cDNA libraries. From the fiber cDNA libraries,about 200 cDNA clones were randomly picked out and subsequentlysequenced. Some clones with potential involvement in cell expansion wereselected according to the sequence data.

To find cDNA clones whose transcripts are specifically expressed incotton fibers, the expression pattern of the selected cDNA clones wasanalyzed by Northern blot hybridization with total RNAs isolated fromcotton fibers, ovules, anthers, petals, sepals, squares, leaves androots, using probes from the clones. RNA samples from the differentcotton tissues were separated on agarose-formaldehyde gels, andtransferred onto Hybond-N nylon membranes by capillary blotting. RNANorthern blots were hybridized in ExpressHyb solution (Clontech) at 68°C. with ³²P cDNA probes prepared by random labeling (PromegaPrime-a-Gene Labeling System). After hybridization, the blots werewashed at 68° C. in 0.1×SSC, 0.5% SDS for 30–60 minutes. Theexperimental results showed that one cDNA clone strongly expressed inyoung fibers of 8 and 14 DPA, and also expressed in young ovules of 4, 8and 14 DPA, but less or not at all in other tissues.

PCR fragments and cDNA fragments were subcloned into vectors, andplasmid DNA prepared with a Qiagen plasmid kit was used as templates inPCR reactions. The PCR products were sequenced by autosequencer.Sequencing of the cDNA clone revealed that it was 1623 bp in lengthcontaining a open reading frame of 1338 bp, and identical to theβ-tubulin gene (FIG. 1). This is the first CFTUB2 cDNA clone isolatedfrom cotton. Comparing the nucleotide and predicted polypeptidesequences of the cotton CFTUB2 with the data banks, it was found thatthe CFTUB2 cDNA shared 96%–98% homology at the amino acid level and over78% homology at the nucleotide level with the known β-tubulin cDNAs andgenes from other plants (such as Arabidopsis, tobacco, rice, soybean,maize, potato, carrot, etc.) [Liaud et al., 1992; Snustad et al., 1992;Villemur et al, 1994; Tonoike, et al., 1994; Taylor et al., 1994; Kanget al., 1994; Okamura et al., 1997; Chu et al., 1998; Okamura et al.,1999].

Total RNAs from different tissues of cotton were used toreverse-transcribe first-strand cDNAs which were used as templates indifferential display PCR reactions. Differential display analysis wascarried out by using a differential display kit (Clontech). First-strandcDNA was synthesized with 2 pg total RNA as starting materials ofreverse transcription and oligo(dT) as primers at 42° C. for 1 hour.Differential display PCR reactions were carried out with a initial cycleconsisting of 94° C. for 5 minutes, 40° C. for 5 minutes and 68° C. for5 minutes, followed by two cycles consisting of 94° C. for 2 minutes and40° C. for 5 minutes and 68° C. for 5 minutes, and then 25 cyclesconsisting of 94° C. for 1 minute and 60° C. for 1 minute and 68° C. for2 minutes, and a final extension at 68° C. for 7 minutes. Targetdifferential display bands were excised and reamplified for furtheranalysis. Reproducible fiber-specific differential display products weretargeted for further analysis. The cDNA in each target band washarvested and regenerated by PCR amplification. The isolated cDNA wassubsequently subcloned into vectors and sequenced.

The Northern blot analysis showed that the transcripts of the CFTUB2gene exhibited a highest accumulation in cotton young fibers of 8 DPA,and then there was a visible decrease in the accumulation of the geneproducts (mRNA) with further development of the fibers. Comparison ofgene expression in different developmental stages of cotton ovules alsoshowed that the gene transcripts accumulated more in 8 DPA ovules thanin 4 and 14 DPA, and there was a gradual and visible decrease to anundetectable level in the accumulation of gene products with fiberdevelopment from 8 DPA to 28 DPA. This suggests that the gene isspecifically expressed with a strict regulation at the transcriptionallevel during cotton fiber and ovule development, as seen with othercotton fiber-specific genes [Whittaker and Triplett, 1999; Shin andBrown, 1999; Kawai et al., 1998; John, 1996a; Song and Allen, 1997; Maet al, 1997; Rinehart et al., 1996; John and Crow, 1992].

EXAMPLE 2 Isolation of the CFTUB2 Promoter

Based on the screened CFTUB2 cDNA sequence, the CFTUB2 promoter wasisolated from cotton Genome Walker libraries by Genome Walker PCR.

Total DNA was extracted and purified from leaves of cotton plants byusing the following method. Liquid N₂ was added to 4 g of leaf tissues,and the leaves were homogenized thoroughly. 20 ml ice-cold extractionbuffer (63 g/L glucose, 0.1 M Tris.HCl (pH 8.0), 5 mM EDTA, 20 g/LPVP-40, 1 g/L DIECA, 1 g/L ascorbic acid, 2 ml/L, betamercaptoethanol)was added to the homogenized tissues in a 50 ml tube and centrifuged at2500 rpm for 15 minutes. After removing the supernatant, 10 ml lysisbuffer was added to each tube. The resuspended pellets were incubated at65° C. for 30 minutes. 10 ml chloroform was added to each tube, mixedwith the samples and centrifuged at 3500 rpm for 10 minutes. Thesupernatant was transferred to a clean tube, and chloroform extractionwas repeated one more time. The supernatant was transferred to a cleantube, and 0.6 volume isopropanol was added to each tube for DNAprecipitation. After centrifuging at 3500 rpm for 30 minutes, the DNAwas washed with 70% ethanol. The isolated genomic DNA was then dissolvedin sterile water or TE (10 mM Tris.HCl, 1 mM EDTA) for use.

Cotton genomic DNA libraries were constructed from leaves of cottonplants. DNA was partially digested with BamH I, and the DNA fragmentswere cloned in the BamH I site of the ZAP expression vector(Stratagene).

Genome Walker libraries were constructed by using Universal GenomeWalker kit (Clontech). Genomic DNA from leaves of cotton plants wasdigested with five restriction enzymes respectively, and then purifiedby phenol/chloroform and precipitated by ethanol. Digested DNA wasligated to Genome Walker adaptors. Two rounds of Genome Walker PCRreactions were carried out successively. 1 μl of each Genome Walker DNAlibrary was used as templates in the primary PCR, and the primary PCRproducts were used as templates in secondary PCR. The PCR was started at95° C. for 1 minute, followed by 35 cycles consisting of 95° C. for 15seconds and 68° C. for 4 minutes, and a final extension at 68° C. for 6minutes. Target PCR bands were cut out and purified by Geneclean kit(Bio 101).

Two fragments in the promoter region were isolated and cloned intopGEM-T vector, respectively. One fragment of the CFTUB2 promoter was1433 bp in length FIG. 2, and another was 984 bp long. FIG. 2. A HindIII site and a BamH I site were created at the 5′-end and 3′-end of the0.9 kb CFTUB2 promoter fragment of cotton respectively by PCR method.The Hind III/BamH I fragment was initially subcloned into pGEM-T vector(Promega). Plasmid DNA containing the CFTUB2 promoter fragment wasdigested with Hind III and BamH I, and the digested fragment wasisolated by agarose gel electrophoresis. A chimeric CFTUB2 promoter/GUSconstruct was generated by insertion of the fragment, replacing CaMV 35Spromoter, into the Hind III/BamH I sites of pBI121 vector.

The 1.3 kb of BamH I/BamH I CFTUB2 promoter fragment was initiallysubcloned into the pGEM-T vector (Promega). Plasmid DNA containing theCFTUB2 promoter fragment was digested with BamH I, and the digestedfragment was isolated by agarose gel electrophoresis. A chimeric CFTUB2promoter/GUS construct was generated by insertion of the fragment intothe BamH I site of pBI101 vector.

EXAMPLE 3 Functional Analysis of the CFTUB2 Promoter

In order to characterize the function of CFTUB2 promoter infiber-specific expression of the CFTUB2 gene, a 1.3 kb of fragment and a0.9 kb of fragment of the CFTUB2 promoter were fused with gus codingsequence in the gene expression vector pBI101 or pBI121 (deletingCaMV35S promoter), respectively (FIG. 3). The constructs of CFTUB2promoter/GUS fusion gene were used to transform tobacco and cotton byAgrobacterium-mediated gene transfer, using the pBI121 vector containingCaMV35S promoter/GUS fusion as a positive control. The CaMV35S promoteris active in all the tissues of cotton and other plants and is aconstitutive promoter [Odell et al., 1985; Ow et al., 1987; McCabe andMartinell, 1993]. A binary vector containing either a CFTUB2promoter/GUS fusion gene or the CaMV35S promoter/GUS control control wastransferred into Agrobacterium tumefaciens strain LBA 4404. Cottonexplants for transformation were obtained from cotton seedlings grown asin Example 1. Tobacco explant material was obtained from tobaccoseedlings. Tobacco seeds were surface-sterilized with 70% ethanol for30–60 seconds and 0.1% HgCl₂ for 15 minutes, followed by washing withsterile water. The seeds germinated on ½ MS medium on light at 28° C. inculture room, and leaves cut from sterile seedlings for use as explantsfor transformation. Cotton cotyledon and hypocotyl explants and tobaccoleaf explants were transformed by the Agrobacterium with the vectors,and transformed plants were transplanted to soil in greenhouse forgrowing to maturity.

Tobacco leaves were cut into about 2×2 cm pieces, and immersed inAgrobacterium suspension for 5 minutes. The infected tobacco explantswere cultivated on MS medium with 1 mg/L 6-BA for 48 hours at 28° C.,and then transferred onto selection MS medium containing 100 mg/Lkanamycin and 1 mg/L 6-BA for 20–30 days for selecting transformedshoots (kanamycin-resistant shoots). The transformed shoots were cutfrom the calli and rooted on MS medium with 50–100 mg/L kanamycin. Thetransformed tobacco plants were transplanted to soil in greenhouse forgrowing to maturity.

The cotyledon and hypocotyl were used as explants for cottontransformation. Cotton seeds were surface-sterilized with 70% ethanolfor 30 seconds and 10% H₂O₂ for 60 minutes, followed by washing withsteril water. These seeds were incubated in the sterile water at 28° C.After over night, the seeds sprouted. The embryos were taken out and puton the IM medium (½ {MS (macronutriants, micronutrients, EDTA-Fe)+VB1 10mg/L+VB6 1 mg/L+VPP 1 mg/L+Myo-Insitol 100 mg/L}+phytogel 2 g/L pH=6.4)at 28° C. for 7 days. The cotyledon and hypocotyl of cotton were used asexplants for transformation. After cutting into 5 mm² (mm) piece, theexplants were soaked in the Agrobacterium tumefaciens strain LBA 4404suspension (OD₆₀₀=0.2–0.4) for 15 minutes. Then the explants were put onCM medium (MS (macronutriants, micronutrients, EDTA-Fe)+VB1 10 mg/L+VB61 mg/L+VPP 1 mg/L+Myo-Instiol 100 mg/L+2.4-D 0.1 mg/L+KT 0.1mg/L+Glucose 30 g/L+MgCl₂ 0.7 mg/L+phytogel 2 g/L pH=6.4) at 24° C. for2 days. After washing with liquid MS medium, the explants were put onthe SM medium (MS (macronutriants, micronutrients, EDTA-Fe)+VB1 10mg/L+VB6 1 mg/L=VPP 1 mg/L+Myo-Insitol 100 mg/L+2.4-D 0.1 mg/L+KT 0.1mg/L+Glucose 30 g/L+MgCl₂ 0.7 mg/L+phytogel 2 g/L+Kanamycin 50mg/L+Cefutoxime 200 mg/L pH=6.4) on light at 28° C. in culture room forselecting and the subculture was per month. After 2–3 monthssubculturing on SM, the calli were induced from explants. The calli weretransferred on DM medium (MS (macronutriants, micronutrients,EDTA-Fe)+VB1 10 mg/L+VB6 1 mg/L+VPP 1 mg/L+Myo-Insitol 100 mg/L+KNO₃ 19g/L+MgCl₂ 0.7 mg/L+Glucose 30 g/L+phytogel 3 g/L pH=6.4) and subculturedper month. After about 5 months, the somatic embryos begin to form.Continuing to culture the young embryos on DM medium until they developinto maturity. The mature embryos were transferred on GM medium (½ {MS(macronutriants, micronutrients, EDTA-Fe)+VB1 10 mg/L+VB6 1 mg/L+VPP 1mg/L+Myo-Insitol 100 mg/L)+NAA 0.01 mg/L+Glucose 30 g/L+phytogel 3.5 g/LpH=6.4) in the box for developing into plantlets. And then the plantletswere transplanted in the soil for the plant growing and collecting thetransgenic seeds.

Transgenic tobacco and cotton plants possessing the chimeric CFTUB2promoter/GUS gene (or ³⁵S:GUS gene), and non-transformed plants asnegative controls, were analyzed by DNA Southern blot hybridization andby GUS histochemical assay. Total genomic DNA from cotton and tobaccoleaves were digested with restriction enzymes, separated on agarosegels, and transferred onto Hybond-N nylon membranes by capillaryblotting. DNA Southern blots were hybridized in ExpressHyb solution(Clontech) at 68° C. with ³²P-DNA probes prepared by random labeling(Promega Prime-a-Gene Labeling System). After hybridization, the blotswere washed at 68° C. in 0.1×SSC, 0.5% SDS for 30–60 minutes. The³²P-labeled nylon membranes were exposed to X-ray film at −70° C. forautoradiography. The results of Southern blot analysis demonstrated thatCFTUB2 promoter/GUS gene was integrated into tobacco and cotton genomes.Total of 325 transformed cotton plants, which belong to 31 transformedlines, were obtained and transplanted in soil to grow to maturation.

Histochemical assays for GUS activity in transgenic tobacco and cottonplants were conducted according to the protocol described previously byJefferson et al. (1987) with some modifications. Fresh tissues from theplants were incubated in X-gluc (5-bromo-4-chloro-3-indolylglucuronide)solution consisting of 0.1 M sodium phosphate (pH 7.0), 10 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM potassium ferrocyanide and 0.5 mMpotassium ferricyanide, and 0.1% X-gluc (Clontech chemical) overnight.The stained plant materials were then cleared and fixed by rinsing with100% and 70% ethanol successively, and the samples were examined andphotographed directly or under a microscope. Consistent with the resultsfrom Northern blot analysis, the GUS gene driven by CFTUB2 promoterspecifically expressed in the young fibers, but not in other tissues, inall the 31 transgenic cotton plants studied, while the GUS activity wasdetected in all the tissues of positive control cotton plants (³⁵S:GUS).A total of 36 transformed cotton plants were obtained and transplantedin soil to grow to maturity, all of which had detectable GUS activityonly in the young fibers, not in the flower organs such as anthers,petals and sepals, or in leaves and roots. Similarly, it was found thatunder the CFTUB2 promoter, GUS gene activity was only detected in theseeds in all of the 15 transgenic tobacco plants studied, suggesting theCFTUB2 promoter activity was also tissue-specific in tobacco. Thisresult, together with the above Northern blot analysis, indicates thatthe CFTUB2 promoter controls gene specific expression at thetranscriptional level in cotton fibers.

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1. An isolated cotton fiber promoter comprising the promoter of thecotton β-tubulin gene CFTUB2 consisting of the sequence of SEQ ID NO:2.2. An isolated cotton fiber promoter comprising the promoter of thecotton β-tubulin gene CFTUB2 consisting of the sequence of nucleotides449–1433 of SEQ ID NO:2.